Abstract

Glioblastoma, the most common primary brain tumor in adults, is invariably fatal despite all current therapies, with a median survival of about 15 months. Glioblastoma contains cancer stem cells or tumor-initiating cells, a subpopulation of cancer cells that are thought to be important in tumor initiation, progression, heterogeneity, recurrence and resistance to therapy, and thus likely critical targets for therapy. We used a mouse syngeneic glioblastoma stem cell (GSC) tumor model with 005 GSCs, isolated from activated H-Ras and Akt induced, Tp53+/- gliomas in C57Bl/6 mice, which recapitulates the hallmarks of human glioblastoma. Immunovirotherapy takes advantage of the natural inflammatory responses to virus infection and oncolytic virus-induced cancer cell death to drive antitumor immunity. Oncolytic herpes simplex viruses (oHSVs) are genetically-engineered to selectively replicate in cancer cells without harming normal tissue. In this study, we test G47∆-mIL12, an oHSV expressing murine IL-12, a critical cytokine involved in adaptive and innate immune responses as well as anti-angiogenesis.

Immune checkpoints, such as co-inhibitory molecules like CTLA-4 and PD-1 with its ligand PD-L1 play a critical role in regulating immune responses and suppressing antitumor immune effector cells. PD-1 and CTLA-4 are expressed predominantly on T-cells, while PD-L1 is also expressed on endothelial and tumor cells. Blocking antibodies to these molecules have been shown to be very effective at reversing tumor-induced immunosuppression. Therefore, we hypothesized that an IL-12 expressing oHSV, which induces antitumor immune responses, should synergize with checkpoint inhibitor antibodies in inhibiting glioblastoma growth.

Mouse 005 GSCs are highly tumorigenic and relatively non-immunogenic, lacking MHC1 expression, with PD-L1 only expressed on a minority of 005 GSCs in vitro. Both MHC1 and PD-L1 can be induced by IFN-γ. Mice with established orthotopic 005 GSC-derived tumors were treated with a single intratumoral injection of G47∆-mIL12 followed by systemic administration of anti-mPD-1, anti-mPD-L1, anti-mCTLA-4, or isotype control antibodies. Single treatments significantly, but modestly, improved survival compared to mock treatment. The combination of G47∆-mIL12 with either of the three checkpoint inhibitors further extended survival. However, the combination of G47∆-mIL12 with 2 checkpoint inhibitors is able to ‘cure’ mice of 005 brain tumors and protect them from tumor rechallenge. This is associated with a large increase in M1-like macrophages and T effector cells and decrease in T regulatory cells. In this representative glioblastoma stem cell model, the combination of oHSV, local IL12 expression, and 2 systemic checkpoint inhibitors was necessary to eliminate established tumors.